442 Laminin alpha 5 in skin microvascular endothelial cell function and angiogenesis

Abstract

Endothelial cell sprouting, including cell attachment and migration, and capillary vessel formation, are critical processes for angiogenesis. These activities are mediated and regulated by extracellular matrix components. Laminin α4 and α5 are two major laminin α chains expressed in human dermal microvascular endothelial cells (HDMECs). In this study, functions of laminin α5 in HDMEC adhesion, migration, capillary tubule formation in angiogenesis were investigated. Using a lentiviral pL1 vector carrying a laminin α5-specific short hairpin RNA (shRNA), laminin α5 expression in stably transduced HDMEC/pL1 cells was suppressed 70% compared to the control cells. Consequently, cell adhesive ability of HDMEC/pL1 cells was remarkably reduced 60% within an hour; and cell mobility was found decreased 83% and 71%, respectively, at 12 and 48 hours in chamber migration assays. In consistence, laminin α5 knockdown also delayed cell migration as observed in an in vitroscratched migration model, in which HDMEC/pL1 cells migrated and covered approximately 50% less area compared to the control. Moreover, depletion of laminin α5 prevented HDMEC/pL1 cells from forming enclosed capillary-like tubular structure in late stage in a three-dimensional model in vitro. In vivo, homozygous laminin α5 knockout (Lα5-/-) skin grafts was characterized with significantly reduced growth of new blood vessels. Interestingly, a concurrent and significant decrease of integrin receptors of αv and β3 has been found correlated to the laminin α5 knockdown, which implied a possible involvement of laminin α5 in integrin αvβ3-dependent angiogenesis. Furthermore, HDMEC/pL1 cells failed FAK activation during cell migration. FAK phosphorylation on tyrosine 397 changed little in HDMEC/pL1 cells while there was markedly increase in control cells. Overall, our findings suggested an important role of laminin α5 in endothelial cell functions in angiogenesis in cell attachment, migration and capillary vessel formation.